The 6-deoxyerythronolide B synthase (DEBS) is the most extensively studied multimodular polyketide synthase (PKS). During the past project period, we have: (i) solved the X-ray crystal structures of major portions of two DEBS modules; (ii) solved the NMR structure of a prototypical acyl carrier protein domain from DEBS; (iii) investigated mechanistic aspects of key steps in polyketide biosynthesis including intermodular chain transfer, chain elongation, extender unit selection and transfer, ?-ketoreduction, dehydration and macrolactonization; (iv) explored the role of protein-protein interactions in establishing the overall architecture and controlling the selectivity of a PKS module; and (v) interrogated the effect of intramolecularity on the rates of individual reactions within the DEBS catalytic cycle. The biosynthetic engineering implications of these and other insights have also been explored and exploited. During the next project period, we propose to continue our fundamental and applied studies on the structure, mechanism and engineering of DEBS. Specifically, we will attempt to solve the X-ray crystal structures of three new protein constructs including: (i) a large fragment consisting of a nearly complete DEBS module; (ii) a fragment of a DEBS module in which the KS and ACP have been crosslinked to each other; and (iii) a stand-alone acyl transferase (Dsz AT) that is exceptionally efficient at acylating PKS modules in trans. We will also investigate the mechanistic basis for: (i) intra- and inter- modular ketosynthase - acyl carrier protein specificity; (ii) efficient transacylation by Dsz AT; and (iii) rate control of the overall DEBS catalytic cycle, and how it is influenced when unnatural extender units are incorporated. Last but not least, to highlight the practical relevance of the above knowledge, we will: (i) elaborate a newly discovered structure-activity relationship for erythromycin that could lead to explorations within a potentially new binding pocket of the eubacterial ribosome; and (ii) attempt to identify a novel lead substance with medicinally relevant activity against apicomplexan parasites. PUBLIC HEALTH RELEVANCE: The enzymatic assembly line responsible for the biosynthesis of the macrocyclic core of erythromycin is the best-studied example of a multimodular polyketide synthase. This enzyme family produces many important antibiotics in nature. The two principal goals of this project are: (i) to obtain a fundamental understanding of the molecular logic of the erythromycin synthase; and (ii) to exploit this knowledge to make new antibiotics.